CD8+ cytotoxic T lymphocytes (CTL) and CD4+ T cells play crucial roles in host defense against human malignancies. However, it is also becoming clearer that neoplastic cells may evade cell-mediated immunity at multiple levels of the effector/target interaction which, consequently, may impact the metastatic process. We have continued to characterize immune effector mechanisms involved in Ag-specific T cell-mediated cytotoxicity of human carcinoma, with emphasis on the role of Fas/Fas ligand (FasL) interactions. If it is a relevant process, it also raises the possibility that the failure of such solid tumor types to undergo Fas-mediated apoptosis may contribute to tumor escape. The specific objectives of these studies were to: (1) examine the role of the Fas/FasL pathway in the mechanism of lysis by Ag-specific CD8+ CTL and the influence of IFN-gamma, as well as anti-neoplastic agents on the modulation of the lytic or apoptotic outcome; (2) compare colon carcinoma cell lines of primary and metastatic origin for potential differences in their lytic response toward Ag-specific CTL, and to correlate the nature of that response with their characterized malignant phenotype; (3) characterize the role of soluble FasL produced by Ag-specific CD4+ T cells in carcinoma immune reactions; and (4) explore the potential interplay between the acquisition of Fas insensitivity and metastatic phenotype. We have developed an in vitro model to compare the lytic sensitivity of a human primary colon carcinoma cell line (SW480) to its metastatic-derived isolate (SW620) to Ag-specific CTL derived from normal or carcinoma-bearing individuals. We have previously identified HLA-A2-restricted, CD8+ T cell epitopes reflecting the ras codon 12 mutation of Gly to Val, as well as MHC class II-restricted CD4+ T cell epitopes reflecting the ras codon 12 mutations of Gly to Cys or Gly to Val in both preclinical and clinical studies. We demonstrated that these CTL lysed the SW480 and SW620 cell lines, which coexpress the naturally occurring Val12 mutation and the HLA-A2 molecule requisite for productive CTL-target recognition. We found that while IFN-gamma was necessary to promote efficient Ag-specific CTL killing, the effector mechanisms employed in lysis of the primary and metastatic cell lines were distinct. IFN-gamma pretreatment rendered SW480 cells sensitive to both Fas-dependent and Fas-independent (perforin) pathways, whereas SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. In addition to cytokine modulation, we have now shown that treatment of SW480 cells with either 5-fluorouracil (5-FU), CPT-11 (irinotecan) or cisplatin (CDDP) led to enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis through Fas-dependent and Fas-independent pathways. In contrast, treatment of SW620 cells with either CPT-11 or CDDP, but not 5-FU, led to enhanced ICAM-1 expression, resulting in Ag-specific CTL killing via Fas-independent mechanisms only. Moreover, the lytic enhancement caused by chemotherapeutic treatment of SW480 and SW620 targets was accompanied by an increase in the activation of caspase-3-like proteases. Taken collectively, these data suggest that: (a) resistance of certain colon carcinoma cells to the Fas pathway may represent unique tumor escape mechanisms in response to Ag-specific T cell attack; (b) IFN-gamma may help to enhance or restore a Fas-sensitive phenotype in certain colon carcinoma cell populations; and (c) certain anti-neoplastic agents may exhibit unique immunoregulatory properties that promote carcinoma cell death through engaging the cytotoxic capabilities of Ag-specific CTL. To further explore the potential association between Fas responsiveness and the malignant/metastatic phenotype, we selected for the outgrowth of Fas-insensitive SW480 cells by in vitro culture with either agonistic anti-Fas mAb or CD4+ T cell-derived sFasL (as a natural source of sFasL) and then compared the selected with the unselected tumor cells for differences in metastatic behavior in vivo, based on lymph node or splenic metastasis in an athymic mouse model. Overall, Fas-insensitive SW480 tumor cells could be derived, which exhibited enhanced malignant tendencies in vivo. Clearly, the destruction of carcinoma cells through cell contact-independent mechanisms, such as sFasL, may be important determinants against neoplastic cells that downregulate MHC/Ag complexes. However, the idea that sFasL may also "select" for carcinoma cells resistant to Fas-mediated apoptosis introduces the hypothesis that the antitumor immune response contributes, albeit inadvertently, to the progression of the neoplastic process. Thus, the objectives of future studies include: (1) to examine the concept and consequences of loss of Fas function on malignant behavior in experimental syngeneic mouse models; and (2) to identify potential molecular defects or inhibitory processes in colon carcinoma cells which correlate with immune resistance to Fas-mediated apoptosis.